Light-regulated Gene Expression in Bacteria : Fundamentals, Advances, and Perspectives

Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits

Blue-light reception through quaternary transitions

Sensory photoreceptors absorb light via their photosensor modules and trigger downstream physiological adaptations via their effector modules. Light reception accordingly depends on precisely orchestrated interactions between these modules, the molecular details of which often remain elusive. Using electron-electron double resonance (ELDOR) spectroscopy and site-directed spin labelling, we chart the structural transitions facilitating blue-light reception in the engineered light-oxygen-voltage (LOV) histidine kinase YF1 which represents a paradigm for numerous natural signal receptors. Structural modelling based on pair-wise distance constraints derived from ELDOR pinpoint light-induced rotation and splaying apart of the two LOV photosensors in the dimeric photoreceptor. Resultant molecular strain likely relaxes as left- handed supercoiling of the coiled-coil linker connecting sensor and effector units. ELDOR data on a photoreceptor variant with an inverted signal response indicate a drastically altered dimer interface but light-induced structural transitions in the linker that are similar to those in YF1. Taken together, we provide mechanistic insight into the signal trajectories of LOV photoreceptors and histidine kinases that inform molecular simulations and the engineering of novel receptors

Cryo-electron microscopy of Arabidopsis thaliana phytochrome A in its Pr state reveals head-to-head homodimeric architecture

Phytochrome photoreceptors regulate vital adaptations of plant development, growth, and physiology depending on the ratio of red and far-red light. The light-triggered Z/E isomerization of a covalently bound bilin chromophore underlies phytochrome photoconversion between the red-absorbing Pr and far-red-absorbing Pfr states. Compared to bacterial phytochromes, the molecular mechanisms of signal propagation to the C-terminal module and its regulation are little understood in plant phytochromes, not least owing to a dearth of structural information. To address this deficit, we studied the Arabidopsis thaliana phytochrome A (AtphyA) at full length by cryo-electron microscopy (cryo-EM). Following heterologous expression in Escherichia coli, we optimized the solvent conditions to overcome protein aggregation and thus obtained photochemically active, near-homogenous AtphyA. We prepared grids for cryo-EM analysis of AtphyA in its Pr state and conducted single-particle analysis. The resulting two-dimensional class averages and the three-dimensional electron density map at 17 Å showed a homodimeric head-to-head assembly of AtphyA. Docking of domain structures into the electron density revealed a separation of the AtphyA homodimer at the junction of its photosensor and effector modules, as reflected in a large void in the middle of map. The overall architecture of AtphyA resembled that of bacterial phytochromes, thus hinting at commonalities in signal transduction and mechanism between these receptors. Our work paves the way toward future studies of the structure, light response, and interactions of full-length phytochromes by cryo-EM

A restraint molecular dynamics and simulated annealing approach for protein homology modeling utilizing mean angles

BACKGROUND: We have developed the program PERMOL for semi-automated homology modeling of proteins. It is based on restrained molecular dynamics using a simulated annealing protocol in torsion angle space. As main restraints defining the optimal local geometry of the structure weighted mean dihedral angles and their standard deviations are used which are calculated with an algorithm described earlier by Döker et al. (1999, BBRC, 257, 348–350). The overall long-range contacts are established via a small number of distance restraints between atoms involved in hydrogen bonds and backbone atoms of conserved residues. Employing the restraints generated by PERMOL three-dimensional structures are obtained using standard molecular dynamics programs such as DYANA or CNS. RESULTS: To test this modeling approach it has been used for predicting the structure of the histidine-containing phosphocarrier protein HPr from E. coli and the structure of the human peroxisome proliferator activated receptor γ (Ppar γ). The divergence between the modeled HPr and the previously determined X-ray structure was comparable to the divergence between the X-ray structure and the published NMR structure. The modeled structure of Ppar γ was also very close to the previously solved X-ray structure with an RMSD of 0.262 nm for the backbone atoms. CONCLUSION: In summary, we present a new method for homology modeling capable of producing high-quality structure models. An advantage of the method is that it can be used in combination with incomplete NMR data to obtain reasonable structure models in accordance with the experimental data

A structural model for the full-length blue light-sensing protein YtvA from Bacillus subtilis, based on EPR spectroscopy

A model for the full-length structure of the blue light-sensing protein YtvA from Bacillus subtilis has been determined by EPR spectroscopy, performed on spin labels selectively inserted at amino acid positions 54, 80, 117 and 179. Our data indicate that YtvA forms a dimer in solution and enable us, based on the known structures of the individual domains and modelling, to propose a three-dimensional model for the full length protein. Most importantly, this includes the YtvA N-terminus that has so far not been identified in any structural model. We show that our data are in agreement with the crystal structure of an engineered LOV-domain protein, YF1, that shows the N-terminus of the protein to be helical and to fold back in between the β-sheets of the two LOV domains, and argue for an identical arrangement in YtvA. While we could not detect any structural changes upon blue-light activation of the protein, this structural model now forms an ideal basis for identifying residues as targets for further spin labelling studies to detect potential conformational changes upon irradiation of the protein